Sze-Looi Song

Molecular phylogeography of Angiostrongylus cantonensis (Nematoda: Angiostrongylidae) and genetic relationships with congeners using cytochrome b gene marker.

Angiostrongylus cantonensis is an important emerging zoonotic parasite causing human eosinophilic meningitis (or meningoencephalitis) in many parts of the world. To-date there is only a single study using mitochondrial cytochrome b (CYTB) gene to determine its genetic structure in eight geographical localities in Thailand. The present study examined the molecular phylogeography of this rat lungworm and its phylogenetic relationship with congeners using CYTB gene marker. A total of 15 CYTB haplotypes was found in 37 sequences from 14 geographical localities (covering north, west, east, central and south regions) in Thailand. These CYTB haplotypes were distinct from those of A. cantonensis for China and Hawaii. In Thailand, some CYTB haplotypes appeared to be confined to specific geographical localities. The partial CYTB DNA nucleotide sequences separated unequivocally the A. cantonensis isolates of Thailand, China and Hawaii as well as the congeners Angiostrongylus malaysiensis, A. costaricensis and Angiostrongylus vasorum, with A. malaysiensis grouped with A. cantonensis and A. costaricensis grouped with A. vasorum. Likewise the congeners of Metastrongylus and Onchocerca genera could also be clearly differentiated. The present study added two new definitive hosts (Bandicota savilei and Rattus losea) and three new localities (Mae Hong Son in the north, Tak in the west, and Phang Nga in the south) for A. malaysiensis in Thailand, indicating its wide occurrence in the country. Three CYTB haplotypes were found in the Thailand samples of A. malaysiensis. In addition to differentiation of congeners, CYTB gene marker could be used for determining the genetic diversity of a given population/taxon.

Complete mitochondrial genome of Bactrocera arecae (Insecta: Tephritidae) by next-generation sequencing and molecular phylogeny of Dacini tribe

The whole mitochondrial genome of the pest fruit fly Bactrocera arecae was obtained from next-generation sequencing of genomic DNA. It had a total length of 15,900 bp, consisting of 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a non-coding region (A + T-rich control region). The control region (952 bp) was flanked by rrnS and trnI genes. The start codons included 6 ATG, 3 ATT and 1 each of ATA, ATC, GTG and TCG. Eight TAA, two TAG, one incomplete TA and two incomplete T stop codons were represented in the protein-coding genes. The cloverleaf structure for trnS1 lacked the D-loop, and that of trnN and trnF lacked the TΨC-loop. Molecular phylogeny based on 13 protein-coding genes was concordant with 37 mitochondrial genes, with B. arecae having closest genetic affinity to B. tryoni. The subgenus Bactrocera of Dacini tribe and the Dacinae subfamily (Dacini and Ceratitidini tribes) were monophyletic. The whole mitogenome of B. arecae will serve as a useful dataset for studying the genetics, systematics and phylogenetic relationships of the many species of Bactrocera genus in particular, and tephritid fruit flies in general.

Complete Mitochondrial Genome of Three Bactrocera Fruit Flies of Subgenus Bactrocera (Diptera: Tephritidae) and Their Phylogenetic Implications

Bactrocera latifrons is a serious pest of solanaceous fruits and Bactrocera umbrosa is a pest of Artocarpus fruits, while Bactrocera melastomatos infests the fruit of Melastomataceae. They are members of the subgenus Bactrocera. We report here the complete mitochondrial genome of these fruit flies determined by next-generation sequencing and their phylogeny with other taxa of the subgenus Bactrocera. The whole mitogenomes of these three species possessed 37 genes namely, 13 protein-coding genes (PCGs), 2 rRNA and 22 tRNA genes. The mitogenome of B. latifrons (15,977 bp) was longer than those of B. melastomatos (15,954 bp) and B. umbrosa (15,898 bp). This difference can be attributed to the size of the intergenic spacers (283 bp in B. latifrons, 261 bp in B. melastomatos, and 211 bp in B. umbrosa). Most of the PCGs in the three species have an identical start codon, except for atp8 (adenosine triphosphate synthase protein 8), which had an ATG instead of GTG in B. umbrosa, whilst the nad3 (NADH dehydrogenase subunit 3) and nad6 (NADH dehydrogenase subunit 6) genes were characterized by an ATC instead of ATT in B. melastomatos. The three species had identical stop codon for the respective PCGs. In B. latifrons and B. melastomatos, the TΨC (thymidine-pseudouridine-cytidine)-loop was absent in trnF (phenylalanine) and DHU (dihydrouracil)-loop was absent in trnS1 (serine S1). In B. umbrosa, trnN (asparagine), trnC (cysteine) and trnF lacked the TψC-loop, while trnS1 lacked the DHU-stem. Molecular phylogeny based on 13 PCGs was in general concordant with 15 mitochondrial genes (13 PCGs and 2 rRNA genes), with B. latifrons and B. umbrosa forming a sister group basal to the other species of the subgenus Bactrocera which was monophyletic. The whole mitogenomes will serve as a useful dataset for studying the genetics, systematics and phylogenetic relationships of the many species of Bactrocera genus in particular, and tephritid fruit flies in general.

Mitochondrial Genome Supports Sibling Species of Angiostrongylus costaricensis (Nematoda: Angiostrongylidae).

Angiostrongylus costaricensis is a zoonotic parasitic nematode that causes abdominal or intestinal angiostrongyliasis in humans. It is endemic to the Americas. Although the mitochondrial genome of the Brazil taxon has been published, there is no available mitochondrial genome data on the Costa Rica taxon. We report here the complete mitochondrial genome of the Costa Rica taxon and its genetic differentiation from the Brazil taxon. The whole mitochondrial genome was obtained from next-generation sequencing of genomic DNA. It had a total length of 13,652 bp, comprising 36 genes (12 protein-coding genes—PCGs, 2 rRNA and 22 tRNA genes) and a control region (A + T rich non-coding region). It is longer than that of the Brazil taxon (13,585 bp). The larger mitogenome size of the Costa Rica taxon is due to the size of the control region as the Brazil taxon has a shorter length (265 bp) than the Costa Rica taxon (318 bp). The size of 6 PCGs and the start codon for ATP6, CYTB and NAD5 genes are different between the Costa Rica and Brazil taxa. Additionally, the two taxa differ in the stop codon of 6 PCGs. Molecular phylogeny based on 12 PCGs was concordant with two rRNA, 22 tRNA and 36 mitochondrial genes. The two taxa have a genetic distance of p = 16.2% based on 12 PCGs, p = 15.3% based on 36 mitochondrial genes, p = 13.1% based on 2 rRNA genes and p = 10.7% based on 22 tRNA genes, indicating status of sibling species. The Costa Rica and Brazil taxa of A. costaricensis are proposed to be accorded specific status as members of a species complex.

Microsatellite markers from expressed sequence tags (ESTs) of seaweeds in differentiating various Gracilaria species

Gracilaria is a red seaweed that has been cultivated worldwide and is commercially used for food, fertilizers, animal fodder, and phycocolloids. However, the high morphological plasticity of seaweeds often leads to the misidentification in the traditional identification of Gracilaria species. Molecular markers are important especially in the correct identification of Gracilaria species with high economic value. Microsatellite markers were developed from the expressed sequence tags of seaweeds deposited at the National Center for Biotechnology Information database and used for differentiating Gracilaria changii collected at various localities and two other Gracilaria species. Out of 33 primer pairs, only one primer pair gave significant results that can distinguish between three different Gracilaria species as well as G. changii from various localities based on the variation in repeated nucleotides. The unweighted pair group method using arithmetic mean dendrogram analysis grouped Gracilaria species into five main clades: (a) G. changii from Batu Besar (Malacca), Sandakan (Sabah), Bintulu (Sarawak), Batu Tengah (Malacca), Gua Tanah (Malacca), Middle Banks (Penang), Sungai (Sg.) Merbok (Kedah), Teluk Pelandok (Negeri Sembilan), Pantai Dickson (Negeri Sembilan), Sg. Kong-Kong (Johore), and Sg. Pulai (Johore); (b) Gracilaria manilaensis from Cebu, Philippines; (c) G. changii from Morib (Selangor); (d) Gracilaria fisheri from Pattani, Thailand; and (e) G. changii from Pantai Dickson (Negeri Sembilan), Gua Tanah (Malacca), Sg. Merbok (Kedah), Sg. Kong-Kong (Johore), and Sg. Pulai (Johore). This result shows that this primer pair was able to distinguish between three different species, which are G. changii from Morib (Malaysia), G. fisheri from Pattani (Thailand), and G. manilaensis from Cebu (Philippines), and also between different genotypes of G. changii. This suggested that the simple sequence repeat primer we developed was suitable for differentiating between different Gracilaria species due to the polymorphisms caused by the variability in the number of tandem repeats.

Complete mitochondrial genome of Angiostrongylus malaysiensis lungworm and molecular phylogeny of Metastrongyloid nematodes

Angiostrongylus malaysiensis is a nematode parasite of various rat species. When first documented in Malaysia, it was referred to as A. cantonensis. Unlike A. cantonensis, the complete mitochondrial genome of A. malaysiensis has not been documented. We report here its complete mitogenome, its differentiation from A. cantonensis, and the phylogenetic relationships with its congeners and other Metastrongyloid taxa. The whole mitogenome of A. malaysiensis had a total length of 13,516bp, comprising 36 genes (12 PCGs, 2 rRNA and 22 tRNA genes) and a control region. It is longer than that of A. cantonensis (13,509bp). Its control region had a long poly T-stretch of 12bp which was not present in A. cantonensis. A. malaysiensis and A. cantonensis had identical start codon for the 12 PCGs, but four PCGs (atp6, cob, nad2, nad6) had different stop codon. The cloverleaf structure for the 22 tRNAs was similar in A. malaysiensis and A. cantonensis except the TΨC-arm was absent in trnV for A. malaysiensis but present in A. cantonensis. The Angiostrongylus genus was monophyletic, with A. malaysiensis and A. cantonensis forming a distinct lineage from that of A. costaricensis and A. vasorum. The genetic distance between A. malaysiensis and A. cantonensis was p=11.9% based on 12 PCGs, p=9.5% based on 2 rRNA genes, and p=11.6% based on 14 mt-genes. The mitogenome will prove useful for studies on phylogenetics and systematics of Angiostrongylus lungworms and other Metastrongyloid nematodes.

Development of chloroplast simple sequence repeats (cpSSRs) for the intraspecific study of Gracilaria tenuistipitata (Gracilariales, Rhodophyta) from different populations

BackgroundGracilaria tenuistipitata is an agarophyte with substantial economic potential because of its high growth rate and tolerance to a wide range of environment factors. This red seaweed is intensively cultured in China for the production of agar and fodder for abalone. Microsatellite markers were developed from the chloroplast genome of G. tenuistipitata var. liui to differentiate G. tenuistipitata obtained from six different localities: four from Peninsular Malaysia, one from Thailand and one from Vietnam. Eighty G. tenuistipitata specimens were analyzed using eight simple sequence repeat (SSR) primer-pairs that we developed for polymerase chain reaction (PCR) amplification.FindingsFive mononucleotide primer-pairs and one trinucleotide primer-pair exhibited monomorphic alleles, whereas the other two primer-pairs separated the G. tenuistipitata specimens into two main clades. G. tenuistipitata from Thailand and Vietnam were grouped into one clade, and the populations from Batu Laut, Middle Banks and Kuah (Malaysia) were grouped into another clade. The combined dataset of these two primer-pairs separated G. tenuistipitata obtained from Kelantan, Malaysia from that obtained from other localities.ConclusionsBased on the variations in repeated nucleotides of microsatellite markers, our results suggested that the populations of G. tenuistipitata were distributed into two main geographical regions: (i) populations in the west coast of Peninsular Malaysia and (ii) populations facing the South China Sea. The correct identification of G. tenuistipitata strains with traits of high economic potential will be advantageous for the mass cultivation of seaweeds.

Complete mitochondrial genome, genetic diversity and molecular phylogeny of Gracilaria salicornia (Rhodophyta: Gracilariaceae)

Abstract: Gracilaria salicornia is an agarophyte that has been used as food and as a source of agar. We sequenced the complete mitogenome of the species from Malaysia and compared it with the previously described sequence from Hawaii. It had a total length of 25,915 bp, comprising 50 genes [25 protein-coding genes (PCGs), 2 rRNA genes and 23 tRNA genes]. It was larger than the Hawaiian sequence (25,272 bp) and possessed three additional tRNAs (trnY, trnR, trnS1). Both G. salicornia sequences had the same start and stop codons for all the PCGs. The genetic distance between the peninsular Malaysian and Hawaiian samples was 0.44% based on 19 PCGs, 0.15% based on 2 rRNA genes and 0.38% based on 19 PCGs + 2 rRNA genes. The phylogram of G. salicornia within Gracilariaceae based on 19 PCGs was congruent with that based on 19 PCGs + 2 rRNA genes.

Cytochrome c oxidase subunit I haplotype reveals high genetic diversity of Angiostrongylus malaysiensis (Nematoda: Angiostrongylidae)

The rat lungworm Angiostrongylus malaysiensis is a metastrongyloid nematode parasite. It has been reported in Malaysia, Thailand, Laos, Myanmar, Indonesia and Japan. In this study, A. malaysiensis adult worms recovered from the lungs of wild rats in different geographical regions/provinces in Thailand were used to determine their haplotype by means of the mitochondrial partial cytochrome c oxidase subunit I (COI) gene sequence. The results revealed high COI haplotype diversity of A. malaysiensis from Thailand. The geographical isolates of A. malaysiensis from Thailand and other countries formed a monophyletic clade distinct from the closely related A. cantonensis. In the present study, five new haplotypes were identified in addition to the four haplotypes reported in the literature. Phylogenetic analysis revealed that four of these five new haplotypes - one from Mae Hong Song (northern region), two from Tak (western region) and one from Phang Nga (southern region) - formed a distinct clade with those from Phatthalung (southern region) and Malaysia. The haplotype from Malaysia was identical to that of Phatthalung (haplotype AM1). In general, the COI sequences did not differentiate unambiguously the various geographical isolates of A. malaysiensis. This study has confirmed the presence of high COI genetic diversity in various geographical isolates of A. malaysiensis. The COI gene sequence will be suitable for studying genetic diversity, population structure and phylogeography.

High Diversity of Bacterial Communities in Developmental Stages of Bactrocera carambolae (Insecta: Tephritidae) Revealed by Illumina MiSeq Sequencing of 16S rRNA Gene

Bactrocera carambolae is a highly polyphagous fruit pest of agricultural importance. This study reports the bacterial communities associated with the developmental stages of B. carambolae. The microbiota of the developmental stages were investigated by targeted 16S rRNA gene (V3–V4 region) sequencing using the Illumina MiSeq. At 97% similarity, there were 19 bacterial phyla and unassigned bacteria, comprising 39 classes, 86 orders, 159 families and 311 genera. The bacterial composition varied among the specimens of developmental stage and across developmental stages as well as exuviae. Four phyla of bacteria (with relative abundance of ≥1% in at least one specimen)—Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria—were recovered from the larva, pupa, adult stages and exuviae. Proteobacteria was the predominant phylum in all the developmental stages as well as the exuviae. Enterobacteriaceae (Proteobacteria) was the predominant family in the adult flies while the family [Weeksellaceae] (Bacteroidetes) was predominant in the larval and pupal stages. Among the genera occurring in more than one developmental stage of B. carambolae, Erwinia was more abundant in the larval stage, Halomonas more abundant in adult female, Stenotrophomonas more abundant in adult male, and Chryseobacterium more abundant in the larval and pupal stages. The results indicate transmission of bacteria OTUs from immatures to the newly emerged adults, and from exuviae to the environment.